The invention relates to novel transcriptional regulatory elements derived from manganese superoxide dismutase (MnSOD) genes, as well as methods of identifying and using the regulatory elements to control gene expression.
Precise control of regulated gene expression has multiple potential applications including inducible gene targeting, overexpression of cytotoxic or cytoprotective genes, antisense RNA expression, and somatic gene therapy (Wettstein et al., 1988).
The ability to produce biologically active polypeptides is increasingly important to the pharmaceutical industry. Over the last decade, advances in biotechnology have led to the production of important proteins and factors from bacteria, yeast, insect cells and from mammalian cell culture. Mammalian cultures have advantages over cultures derived from the less advanced life forms in their ability to post-translationally process complex protein structures such as disulfide-dependent folding and glycosylation. Neuroendocrine cell types have added unique capacities of endoproteolytic cleaving, C-terminal amidation and regulated secretion. Indeed, mammalian cell culture is now a preferred source of a number of important proteins for use in human and animal medicine, especially those which are relatively large, complex or glycosylated. Improved methods for expressing desirable polypeptides in mammalian host cells are highly desirable.
Gene therapy involves the transfer of one or more functional homologous genes, and the sequences controlling their expression, into a target cell. The purpose of gene therapy is to replace a defective or deficient gene, the absence of which produces a pathological state or to supplement an endogenous gene product to achieve a therapeutic effect (Berns and Giraud, 1995). Viral vectors are widely used vehicles for the effective delivery of genes into mammalian cells which have the capability to infect high proportions of cells in a cell population (Friedmann and Yee, 1995; Friedmann, 1997). Some of the best examples of viral gene targeting vectors are based on retroviral, adeno (Ad) or adeno-associated (AAV) viruses. However, vectors developed from these viruses all lack some level of specificity which presents an obstacle for appropriate and controlled expression of foreign genes (Friedmann, 1996). For example, retroviruses are generally limited to transduction of dividing cells whereas Ad and AAV can transduce non-dividing cells. On the other hand, repeated administration of recombinant Ad based vectors, is often limited by host immune responses against viral structural proteins. Presently, AAV may hold the greatest promise in that rAAV does not appear to induce an inflammatory or immune response, and is only limited by the inability to easily produce high rAAV virion titers.
AAV is a single stranded human DNA virus with a genome length of 4.7 kb (Muzyczka, 1992; Srivastava et al., 1983; Yang and Trempe, 1993) that requires a helper virus for productive growth. Adeno (Ad) or herpes virus family members can provide helper function in established human tissue culture lines, whereas only adenovirus is found associated with AAV in human isolates. The normal route of infection for AAV is via the respiratory or intestinal tracts analogous to Ad. If a helper virus is not available during AAV infection, the AAV genome integrates into a human chromosome 19 and is propagated as a stable provirus. Superinfection with Ad leads to AAV provirus excision and a normal productive growth cycle that results in the production of a mixed viral stock consisting of both wild type AAV and Ad virus particles. AAV possesses unique biological properties, which has led to its exploitation as a versatile gene therapy vector. Most notably AAV undergoes a latency phase, which often involves stable integration within a region of chromosome 19 known as the AAVS1 site, thus establishing a persistent infection with very little host response (Cheung et al., 1980; Conrad et al., 1996). Perhaps the most attractive AAV feature is that even though human exposure to this virus is commonplace, no diseases has been associated with AAV infections in either animal or human populations (Blacklow et al., 1967; Blacklow et al., 1968, Blacklow et al., 1971; Hoggan, 1970), nor have there been any reports of rAAV induced inflammatory responses. In addition, it has been demonstrated that recombinant AAV (rAAV) vectors do not integrate in lung epithelial cells (Flotte et al., 1993; Afione et al., 1996), rAAV persists for up to six months potentially as an unintegrated episome.
Numerous AAV vectors have been developed to exploit the latency pathway, where, in general, vectors are generated by deleting the viral coding sequences and substituting the appropriate transgene controlled by a promoter and flanked by the AAV-TRs. By keeping the construct size at xcx9c5 kb which is within the packaging limit, these vectors can be incorporated into infectious virions in trans in Ad-infected cells. Such recombinant virions have been found to infect a variety of cell types in vitro and in vivo including hematopoietic cells (Goodman et al., 1994) neurons (Kaplitt et al., 1994), airway epithelial cells (Flotte et al., 1992), as well as skeletal and cardiac muscle (Kessler et al., 1996).
One of the main issues in potential clinical application of gene therapy is the need for increased gene transfer efficiency and target specificity associated with regulated expression at therapeutically relevant levels in vivo (Chow et al., 1997). Effective gene therapy, therefore, must include the design of crucial regulatory elements, promoters and enhancers, which possess cell type specific activities and can be activated by certain physiologically relevant induction factors (e.g., hormones, cytokines, chemokines, irradiation, heat shock) via responsive elements. Controlled and restricted expression can be achieved using such regulatory elements to drive the expression of therapeutic genes in plasmid based as well as viral vector constructs (Hwang et al., 1997; Sandig and Strauss, 1996; Finke et al., 1998). In addition to high level and efficient gene expression, minimizing or excluding inappropriate gene expression in surrounding non-target cells will be of great importance for numerous gene therapy applications (Namba et al., 1998; Hesdorffer et al., 1998; Gossen and Bujard, 1992).
Unfortunately, almost all of the presently available inducible promoters used in gene therapy vectors require exogenous stimulation by non-physiological or artificial substances such as tetracycline (No et al., 1996), ecdysone (Delort and Capecchi, 1996), or RU486 (Massie et al., 1998). One of the disadvantages of these systems is that they all require expression of two gene products. Namely, the expression of the desired transgene driven by an inducible promoter which requires the expression of a non-endogenous receptor. This is usually accomplished by co-transfection of separate plasmids into mammalian cells, thus potentially limiting the size of the transgene when a viral vector system is used for transgene delivery. In addition, to maintain high levels of expression with the aforementioned inducible promoters, the exogenous substance must be continuously supplied. The disadvantages of these systems include the continuous treatment with the exogenous substance and slow clearance from the organism, which interferes with quick and precise induction. Alternatively high levels of constitutive expression can be obtained with the cytomegalovirus (CMV) promoter in many cell types, which will be beneficial in certain disease states, where an example might be the CFTR gene product for cystic fibrosis.
It has been suggested that the enhancer elements of mammalian genes might allow the desired precise control of gene regulation needed for transgene expression (Maxwell et al., 1996; Clesham et al., 1996; Walther and Stein, 1996; Hofmann et al., 1996; Raoul et al., 1998). Such an enhancer element should be tissue-specific and stimulant-specific, allowing the transgene expression to be kept at a low basal level. However, such an element should allow dramatic induction in response to the precise stimulus. Ideally, the stimulus causing this induction could be endogenously produced and potentially associated with the disease pathology. Most intracellular trans-activators of enhancer elements are typically stimulated as part of signal transduction pathways from transmembrane or intracellular receptors responding to extracellular ligands (hormones, cytokines, chemokines). Therefore, unlike the presently available inducible promoters systems, an ideal regulatory sequence would function through an endogenous stimulus, receptor and signal transduction pathway.
Many diseases amenable to gene therapy will not require continuous high constitutive gene expression since the disease state itself is episodic in nature (inflammatory or ischemic diseases). In these situations, low levels of gene expression during the normal state are adequate, but the capacity to increase expression during the onset of inflammation or ischemia would be advantageous for the precise control sought. High level expression of cytoprotective proteins (such as MnSOD. Hohmeier et al., 1998; Majima et al., 1998; Epperly et al., 1998) during inflammation/ischemia of various organ systems (lungs, brain, small and large intestine) would either halt or slow progression of the disease process (Waxman et al., 1998; Manna et al., 1998; Arai et al., 1990). However, available inducible promoters do not allow the cell to control the timing of expression of the transgene or its fold induction. An ideal inducible clement would make use of the cell or tissues own signaling system to increase expression of the transgene of interest. Under conditions of inflammation, the most precise inducible element would probably utilize the cytokine systems.